Carboxypeptidases catalyze the removal of the C-terminal amino acid residues in peptides and proteins. Carboxypeptidases have important biological functions and are widely distributed. Carboxypeptidases are found in the pancreas and digestive tract (carboxypeptidases A and B, CPA and CPB), mast cells (mast cell carboxypeptidase A), the secretory granules of endocrine and neuroendocrine cells (carboxypeptidases E and D, CPE and CPD), plasma cell membrane (carboxypeptidase M, CPM) and the blood (carboxypeptidase N, CPN). Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) is a plasma zymogen of a basic carboxypeptidase. Activated TAFI (TAFIa), also called carboxypeptidase U, plasma carboxypeptidase B and carboxypeptidase R, removes C-terminal Arg or Lys residues.
Removing a C-terminal amino acid may dramatically affect biological functions of peptides and proteins. For example, TAFIa regulates fibrinolysis by removing C-terminal Arg or Lys residues. The fibrinolytic cascade is the physiological pathway for plasmin activation and fibrin degradation. Partially degraded fibrin contains C-terminal basic residues that accelerate fibrinolysis by providing binding sites for tissue plasminogen activator (tPA), plasminogen and plasmin. Exposed C-terminal lysine residues of partially degraded fibrin plays a key cofactor role by accelerating the rate of plasmin activation by two to three orders of magnitude. Thus, TAFIa curtails fibrinolysis by removing C-terminal lysine residues. TAFIa inhibitors would therefore be beneficial in the treatment of thrombosis.
TAFIa is unstable at physiological temperature and activity quickly diminishes during serum preparation. Hendriks, D., Scharpe, S., van Sabde, M, and Lommaert, M. P. (1989), J. Clin. Chem. Clin. Biochem. 27, 277–285. TAFIa shows a half-life of 74 min at 25° C. Mao, S-S., Cooper, C. M., Wood, T., Shafer, J. A. and Gardell, S. J. (1999), J. Biol. Chem. 274, 35046–35052. Although TAFIa is more stable at lower temperature, attempts to efficiently isolate TAFIa at low temperatures were unsuccessful, with <1% activity yield. Wang, W. Hendriks, D. F. and Scharpe, S. S. (1994), J. Biol. Chem. 269, 15937–15944; Hendriks, D., Wang, W., Scharpe, S., Lommaert, M-P., van Savde, M. (1990), Biochim. Biophy. Acta 1034, 86–92. Reversible TAFIa inhibitors such as ε-aminocaproic acid, when added to TAFIa in vitro, have been reported to preserve stability. However, stable TAFIa has not been formed and purified in the presence of a reversible TAFIa inhibitor. Boffa, M. B., Wang, W., Bazjar, L., and Nesheim, M. E. (1998), J. Biol. Chem. 273, 2127–2135;
The present invention provides stable purified activated TAFI, and a method of purifying stable activated TAFI from serum.
The invention also provides more efficient and sensitive assays for carboxypeptidase activity.